Should I dilute cDNA for real-time PCR? - (Feb/06/2006 )
Hi all ...
I'm in the process of performing "real time RT-PCR", I'm just a beginner to this field. What I did so far was I took 4 diffrent samples from the same tissue, same species. After total RNA extraction, I used 6ug to make first-strand cDNA's and stored it at -20 C.
Q: Am I supposed to dilute the cDNA concentration to (e.g 100ng/ul) before I run the real time RT-PCR if my aim was to compare the gene expression of these 4 diffrent samples? Whats the reasoning behind this, if its true?
I did use 2ul of 100ng/ul and ran a RT-PCR to test the specificity of this sample and the band size and intensity was good after 30 cycles.
Any suggestions would be highly appreciated.
Real time pcr is very sensative. Less is best in this case.
I typically use 1ug of total RNA in the cDNA reaction (20ul volume) then dilute 1:10 with depc water.
I will use 5 ul of that in a 25 ul real time pcr reaction.
Here is what you want to do:
Dilute the product from your reverse transcription reaction at least five or ten-fold to remove the PCR inhibitory effects of RT. If you need to use this sample many times and a ten-fold dilution is insufficient, you can go up to a hundred fold dilution without influencing your results deleteriously.
Pipette one to two microliters from your diluted cDNA sample into each 25 ul reaction volume. I personally use two microliters because I don't always have a one microliter pipettor handy, but one microliter is more than enough.
You also need to define a standard for quantifying between samples. Beta-actin is a good one, as is GAPDH (Beta actin is better, though). Order primers for this standard and create a vector containing the cloned standard for easy quantification.
i'm talking about this in another thread, i've been doing real-time pcr for 2 years and didn't know this! no one [including the guy from biorad who trained me] told me you had to dilute the cDAN. with undiluted cDNA most of the primers i have designed have worked, and the primer efficiencies are bang on 95-100%.... weird... first time i diluted my cDNA i got a primer efficiency of 120%!!!!